Endothelial Dysfunction in Hypertension Is Independent From the Etiology and From Vascular Structure
The aim of our study was to evaluate the relationships between endothelial function, small resistance artery structure, and blood pressure in patients with primary or secondary hypertension. Sixty subjects were included in the study: 9 patients with pheochromocytoma, 10 with primary aldosteronism, 17 with renovascular hypertension, and 13 with essential hypertension with 11 normotensive subjects who served as controls. Clinic and 24-hour ambulatory blood pressure (ABPM) were evaluated. All subjects were submitted to a biopsy of subcutaneous fat. Small resistance arteries were dissected and mounted on a micromyograph and the media/lumen ratio was calculated. A dose-response curve to acetylcholine was performed at cumulative concentrations from 10−9 to 10−5 mol/L. The vasodilator response to acetylcholine was similarly impaired in the four groups of hypertensive patients (ANOVA P<.05 versus normotensive controls), without any significant difference among them. In subcutaneous small arteries of patients with either primary aldosteronism or renovascular hypertension, a marked increase in media:lumen ratio was observed, while in patients with pheochromocytoma, the extent of vascular structural alterations was similar to that observed in essential hypertension. No significant correlation between media-lumen ratio or clinic blood pressure and maximum acetylcholine-induced vasodilatation was observed. On the contrary, a significant, albeit not very close, correlation between ABPM values and maximum acetylcholine-induced vasodilatation was observed (r=34, P<.05 with 24-hour systolic blood pressure, r=0.36, P<.05 with 24-hour diastolic blood pressure). In conclusion, endothelial dysfunction seems to be independent from the degree of vascular structural alterations and from the etiology of hypertension, and it is probably more linked to the hemodynamic load.
- ABPM = ambulatory blood pressure monitoring
- ANOVA = analysis of variance
- EH = essential hypertensive patients
- NT = normotensive subjects
- DBP = diastolic blood pressure
- ID = normalized internal diameter
- MCSA = media cross-sectional area
- M/L = media/lumen ratio
- MT = media thickness
- PA = patients with primary aldosteronism
- Phaeo = patients with pheochromocytoma
- RVH = patients with renovascular hypertension
- SBP = indicates systolic blood pressure
- SHR = spontaneously hypertensive rats.
- WT = wall thickness
Endothelial cells are known to have important regulatory effects on the cardiovascular system through the release of vasodilator and vasoconstrictor mediators.1,2⇓ In small resistance arteries, endothelium seems to have a key role in the imbalance between vasoconstriction and vasodilatation.2 An impairment of the endothelial function, as evaluated by the vasodilator response to acetylcholine, has been detected in human small resistance arteries both in essential3,4⇓ and in secondary hypertension.4,5⇓
Structural abnormalities of the media of the resistance vessels are common accompaniments of chronic hypertension, and they play an important role in the increase of vascular resistance, and, therefore, in the maintenance of high blood pressure values.6,7⇓ We have previously demonstrated that the extent and the characteristics of structural alterations observed in subcutaneous small resistance arteries of patients with primary or secondary forms of hypertension are not uniform.4 In particular, in patients with either primary aldosteronism or renovascular hypertension, we observed a marked increase in media:lumen ratio, while in patients with pheochromocytoma, the extent of vascular structural alterations was similar to that present in essential hypertension. The increase in media:lumen ratio in patients with essential hypertension and with pheo-chromocytoma was mainly due to vascular remodeling (rearrangement of the same material around a narrowed lumen), while in patients with renovascular hypertension, there was vascular growth (cell hypertrophy or hyperplasia).4 Patients with primary aldosteronism had an intermediate pattern compared with the other two forms of secondary hypertension.4
Therefore, it is possible that vascular structural alterations may be related to endothelial dysfunction, by directly influencing endothelium-dependent vasodilator responses in hypertension. However, several studies performed in spontaneously hypertensive rats (SHR) have suggested that a hemodynamic rather than a structural factor is involved in the genesis of endothelial dysfunction.8–11⇓⇓⇓ In particular, a significant correlation between systolic blood pressure and maximum acetylcholine-induced vasodilatation was observed in treated and untreated SHR.11 Moreover, endothelial dysfunction seems to be absent in young SHR, before the development of hypertension, despite the presence of early vascular structural alterations;8 similar dissociations may be observed in SHR treated with various drugs.9,10,11⇓⇓
The aim of our study was to evaluate the relationships between endothelial function, small resistance artery structure, and blood pressure in patients with primary or secondary hypertension.
Patients and Methods
Thirty-six patients with secondary hypertension were included in the study: 9 patients with pheochromocytoma, 10 with primary aldosteronism, and 17 with renovascular hypertension. Data were compared with those obtained in 11 normotensive subjects and in 13 patients with essential hypertension.
Hypertensive patients had a clinic blood pressure (the average of three different sphygmomanometric measurements, each performed on three separate days, after a wash-out period of at least 2 weeks if previously treated with antihypertensive drugs) greater than 140/ 90 mm Hg. Normotensive control subjects had a systolic blood pressure lower than 140 mm Hg, and a diastolic blood pressure lower than 90 mm Hg. All hypertensive patients had been previously treated for various periods of time with calcium channel blockers, angiotensin-converting-enzyme inhibitors, diuretics, or β-blockers. There was no statistically significant difference in the therapeutic regimens of the different groups.
In 6 patients with pheochromocytoma, 10 with primary aldosteronism, 10 with renovascular hypertension, 13 with essential hypertension, and 7 normotensive controls, a noninvasive 24-hour blood pressure monitoring (ABPM) (Spacelab 90209, Spacelabs Inc) was performed at the end of the wash-out period, when necessary. Patients were fitted with the ABPM on the morning of the day of the recording. The interval between recordings was 20 minutes. (For further details about the method, see reference 12.)
Venous blood samples were taken with the participants in the supine position, after a wash out period of at least 2 weeks, for measurement of plasma renin activity (RIA, Renctk, Sorin Biomedica), plasma aldosterone (RIA, Aldoctk, Sorin Biomedica) as well as urinary norepinephrine and epinephrine (HPLC).13
The diagnosis of secondary forms of hypertension was made on the basis of an indication for renal artery revascularization (n=17) or adrenal tumor resection (n=17), after proper investigation by imaging techniques and humoral assessments. In one case, a bilateral renal artery stenosis was demonstrated by angiography, but the patient refused intervention. In two cases, a bilateral adrenal hyperplasia associated with high plasma and urinary aldosterone levels was demonstrated. In these cases no surgical correction was performed. Thirty-four patients were submitted to surgery or renal artery angioplasty and had their hypertension cured except for 4 patients with renovascular hypertension in whom blood pressure was significantly reduced, but a antihypertensive monotherapy was still required after 6 months. All these patients were previously placed on a combination therapy (3 or 4 drugs). Essential hypertension was diagnosed on the basis of persistently elevated levels of blood pressure, after a careful exclusion of a secondary cause.
All participants underwent a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. The biopsy of the abdominal subcutaneous fat was taken during a surgical procedure (usually a cholecystectomy in normotensives and essential hypertensives, an adrenalectomy or a vascular surgical intervention on the renal arteries in patients with secondary hypertension), whereas, in the remaining cases, a standard skin biopsy of the gluteal region (3 cm long, 0.5 cm wide, 1.5 cm deep) was performed.14,15⇓ The percentage of skin biopsies taken from abdomen was similar in the five groups of subjects (about 20 to 25%).
We have previously demonstrated that no difference in the morphologic or functional properties of the small resistance arteries obtained from the subcutaneous fat taken from the gluteal (local anesthesia), or from the anterior abdominal region (general anesthesia) could be observed.4
The protocol of the study was approved by the ethics committee of our institution (Medical School, University of Brescia), and informed consent was obtained from each participant. The procedures followed were in accordance with institutional guidelines.
Small arteries (about 160 to 280 μm of average diameter in relaxed conditions, 2 mm long) were dissected from the subcutaneous fat of the biopsies and mounted as a ring preparation on an isometric myograph (410 A, JP Trading), by threading onto two stainless steel wires (40 μm diameter). The wires were attached to a force transducer and micrometer, respectively, as previously described by Mulvany and coworkers.16,17⇓ Vessels were warmed to 37°C and allowed to equilibrate for at least 30 minutes in physiological saline solution (PSS) with the following composition (in mmol/L): NaCl 119, NaHCO3 24, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 2.5, and glucose 5.5, kept constantly at 37°C and bubbled with 5% CO2 in O2. The vessel internal circumference was set to give a wall tension of 0.1 mN/mm.
Vessel wall and media thicknesses were measured at 12 sites, which were then averaged, using a light microscope with immersion lens (Laboratory 20, Carl Zeiss S.p.A) at ×600 magnification, which provides a resolution of 0.2 μm. Lower magnification was used for measurement of the distance between the wires and length of the blood vessel. The resting tension-internal circumference relation was determined, and vessels were set to the normalized circumference L1, where L1=0.9 L100 and L100= is the internal circumference the vessels would have had in vivo, when relaxed and under a transmural of 100 mm Hg. as described previously by Mulvany et al.16,17⇓ From L1, the normalized internal diameter l1 was calculated. Assuming that the cross-sectional area remains constant when the vessel is extended to L1, the wall and media thickness were automatically calculated also in normalized condition. Wall and media thickness as well as the media:lumen ratio of blood vessels in normalized condition (vessels extended L1) were obtained assuming a constant wall and media volume, from wall and media cross-sectional area calculated from wall and media thickness measured in unstretched vessels, as previously described.16,17⇓
The vessels were then stimulated as follows
Three stimulations (2 minutes for each) with PSS in which NaCl was substituted with KCl on an equimolar basis (K-PSS), and two stimulations with K-PSS containing 10 μmol/L norepinephrine;
A cumulative dose-response curve to acetylcholine at the following concentrations: 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μmol/L, 3 minutes per concentration, after precontraction with norepinephrine 5 μmol/L (endothelium-dependent vasodilatation).
In a small subset of subjects (4 normotensives, 4 essential hypertensives, 3 patients with pheochromocytoma, 4 with primary aldosteronisms, and 8 with renovascular hypertension) a dose-response curve to sodium nitroprusside (10−9, 10−8, 10−7, 10−6, 10−5 mol/L) was also performed (endothelium-independent vasodilatation).
The log EC50 (the logarithm of the median effective concentration) was calculated from the dose-response curve to acetylcholine.
The responses of blood vessels are expressed as wall tension (active force divided by two times the segment length). The response to acetylcholine and sodium nitroprusside were expressed as the percentage of decrease of the wall tension obtained with norepinephrine precontraction.
If the vessels produced rhythmic activity, the response was measured from the mean active force for the last 20 seconds of each period.
All chemicals were dissolved in PSS, except sodium nitroprusside (dissolved in glucosate solution and protected from light by aluminum foil). All drugs were obtained from Sigma.
All data are expressed as mean±SD, unless otherwise stated. All important variables were normally distributed. One-way analysis of variance (ANOVA) and Bonferroni’s correction for multiple comparisons were used to evaluate differences among groups. Relationships between endothelial function and vascular structure or blood pressure were evaluated by correlation analysis. Patients with primary and secondary hypertension were considered as a single group, while normotensive subjects have been excluded from the analysis, to avoid a calculation bias, due to an a priori selection of subjects on the basis of their blood pressure. Two-way ANOVA for repeated measures was used for dose-response curves to acetylcholine and sodium nitroprusside (group × dose) (BMDP Statistical Software programs 3 D, 7D, 1V and 2V, BMDP Statistical Software Inc).
No difference in age, gender, height, weight, serum cholesterol or triglycerides among the different groups was observed (Table 1). The duration of hypertension as well as the duration of the previous antihypertensive treatment was similar in the four groups of hypertensive patients. There was no significant difference in the clinical, morphologic, and functional parameters between subjects who perform and those who did not perform ABPM.
In all groups of hypertensive patients clinic systolic and diastolic blood pressure during therapeutic wash-out was significantly increased compared with normotensive subjects, and no significant difference was observed among the hypertensive groups (Table 2).
Similar results were obtained for 24-hour, daytime and nighttime systolic and diastolic blood pressure (Table 2).
The clinic blood pressure values recorded during antihypertensive therapy were similar in the four groups of hypertensive patients (data not shown).
A statistically significant increase in urinary catecholamines was observed in patients with pheochromocytoma, compared with the other groups (Table 2). In addition, plasma renin activity was increased in patients with renovascular hypertension and was reduced in patients with primary aldosteronism, compared with the remaining groups (Table 2). Plasma aldosterone was significantly increased in patients with primary aldosteronism, and with renovascular hypertension.
Media:lumen ratio was significantly increased in essential hypertension and in patients with pheochromocytoma, compared with normotensive subjects. In patients with primary aldosteronism and renovascular hypertension, an even more pronounced increase of media:lumen ratio was observed; in addition, the difference between patients with pheochromocytoma and with essential hypertension was statistically significant. (Table 3). A similar pattern may be observed for the media thickness, although, in this case, no difference was be observed between patients with primary aldosteronism and patients with pheochromocytoma or essential hypertension. The total wall thickness was significantly increased in patients with essential hypertension, primary aldosteronism, and renovascular hypertension, compared with normotensive subjects. In patients with pheochromocytoma, the increase in total wall thickness, compared with normotensive subjects was statistically not significant.
No significant difference among the different groups in the response to KPSS or in the precontraction with norepinephrine was observed (Table 4). The response to acetylcholine patients with essential hypertension, pheochromocytoma, primary aldosteronism, and renovascular hypertension was significantly reduced (ANOVA P<.05 at least in each case) compared with normotensive subjects (Table 4, Fig 1). No difference was observed among the different groups of patients with primary or secondary hypertension. No significant difference among the groups in the sensitivity to acetylcholine, as expressed by the EC50, was observed (normotensive subjects: −7.88±1.29, pheochromocitoma: −7.21±1.31, primary aldosteronism: −7.45±1.02, renovascular hypertension: −7.28±1.06, essential hypertension, −7.45±1.29).
In the small number of subjects in whom a dose-response curve to nitroprusside was performed, no difference in the endothelium-independent relaxation was observed among the groups (Fig 2).
Relationships Between Endothelial Function and Vascular Structure or Blood Pressure
No significant correlation between media-lumen ratio or clinic blood pressure and maximum vasodilatation induced by acetylcholine was observed (Table 5) in the four groups of hypertensive patients. On the contrary, a significant, albeit not very close, correlation between ABPM values and maximum acetylcholine-induced vasodilatation was observed (r=34, P<.05 with 24-hour systolic blood pressure, r=0.36, P<.01 with 24-hour diastolic blood pressure) (Table 5, Fig 3). The correlation coefficients for daytime or nighttime blood pressure values were similar (Table 5). No significant correlation was observed between ED50 of the response to acetylcholine and 24-hour or clinic blood pressure, or indexes of vascular structure.
In the small number of hypertensive patients in whom a dose-response curve to nitroprusside was performed, no significant correlation between maximum nitroprusside-induced vasodilatation and 24-hour blood pressure was observed (data not shown).
No significant correlation between night-time decline in systolic or diastolic blood pressure and indexes of endothelial function was observed.
It is presently unclear whether endothelial dysfunction is a primary abnormality or a consequence of the elevated blood pressure values. Data in animal models of genetic hypertension are in favor of a direct damage of the endothelial cells secondary to a prolonged increase of the hemodynamic load and of the shear stress.8–11⇓⇓⇓ In patients with essential hypertension, primary aldosteronism, or renovascular hypertension,4,5⇓ and also in normotensive offspring of hypertensive parents,18 an impairment of the vasodilator response to acetylcholine in the forearm represents a frequent finding.
For the first time, this study has examined the relation between endothelial function and small resistance artery structure or blood pressure in humans, using direct, reliable, and well assessed techniques. An evident impairment of endothelial function, as evaluated with the dose-response curve to acetylcholine, was observed in all groups of hypertensive patients, regardless of the etiology of hypertension. In addition, the extent of the impairment of endothelial function was similar in patients with primary or secondary hypertension. No relationship between maximum acetylcholine-induced vasodilatation in subcutaneous small resistance arteries and indexes of vascular structure was observed. Thus, our data support the hypothesis that, in humans, endothelial dysfunction seems to be independent from the etiology of hypertension and from the degree of vascular structural alterations.
In our study, endothelial function was evaluated by a vasodilator response to acetylcholine. It is well known that stimulation of muscarinic receptors is associated with the release of endothelium-derived relaxing factor, namely nitric oxide; therefore, the vasodilator response to acetylcholine may be a useful tool to evaluate the endothelial function.2 In some subjects, endothelium-independent vasodilation was also evaluated. No difference in the vascular response to sodium nitroprusside was observed, thus suggesting a specific impairment of synthesis, or release, or of biological activity of endothelium-derived relaxing factors.
It is well known that dyslipidaemia can affect endothelial function; on the other hand, the lipid profile was within normal limits in all subjects, and no difference in serum cholesterol or triglycerides was observed among the different groups.
In our study a significant, albeit not very close, correlation between maximum acetylcholine-induced vasodilatation and 24-hour systolic or diastolic blood pressure (as well as daytime or nighttime values) was observed, thus suggesting that hemodynamic factors are important in the genesis and/or maintenance of endothelial dysfunction. On the contrary, no significant correlation was observed between maximum acetylcholine-induced vasodilation and clinic blood pressure, thus suggesting that ABPM values are better indicators of the total hemodynamic load than isolated, clinic blood pressure measurements. It was previously demonstrated that ABPM values are superior to clinic blood pressure from a prognostic point of view; in fact, they are more closely correlated to target organ damage and to changes in left ventricular mass during treatment.12,19,20⇓⇓ Our findings are supported by recent findings by Forte et al,21 who have observed a significant inverse correlation between average mean daytime ambulatory blood pressure and urinary nitrate excretion in essential hypertensive patients.
However, evidence from other studies appeared to be against the hypothesis that blood pressure may be an important determinant of endothelial dysfunction. In a study performed in isolated aortic rings of SHR, antihypertensive treatment with cilazapril improved endothelial function, while no improvement was observed after hydralazine treatment, despite a similar blood pressure reduction.22 Schiffrin et al23 were able to observe an improved relaxation to 10 μmol/L acetylcholine of subcutaneous small resistance arteries in humans after 1 and 2 years of treatment with cilazapril. No change was observed in patients treated with atenolol, although blood pressure reductions during therapy were comparable. Similar results have been obtained comparing the effects of atenolol and nifedipine in a slow-release formulation.24 Again, a significant improvement of endothelial function was observed after 1 year of treatment with nifedipine, while no change was observed in patients treated with atenolol, despite similar blood pressure reduction. These results suggest that the improvement of the endothelial function observed during treatment could not be explained simply by blood pressure reduction. It is possible that the mechanisms responsible for the genesis and of the regression of endothelial dysfunction may be different, at least in part. The increase in shear stress consequent to blood pressure elevation may be crucial in the induction of the impairement of endothelium-dependent vasodilatation; however, once endothelial dysfunction is established, its regression may involve more complex pathways, and additional beneficial effect on the vessels beyond blood pressure reduction could be required. Further investigation are needed in order to clarify whether abnormalities in endothelial function are results of hypertension or contributing factors to its pathogenesis.25
However, our data support the hypothesis that, in humans, endothelial dysfunction seems to be independent from the etiology of hypertension and from the degree of vascular structural alterations, while it is probably more linked to the hemodynamic load.
The authors thank Antonio Salvetti MD, (Pisa, Italy) and Achille C. Pessina, MD, (Padova, Italy) for precious collaboration, and Miss Alessandra Panarotto for technical assistance.
- Received September 17, 1997.
- Revision received October 10, 1997.
- Accepted October 22, 1997.
Lüscher TF, Vanhoutte PM. The Endothelium: Modulator of Cardiovascular Function. Boca Raton, Florida: CRC Press; 1990: 1 –215.
Lüscher TF, Tschudi MR, Dohi Y. Resistance arteries in hypertension: role of the endothelium. In: Mulvany MJ, Aalkjaer C, Haegerty AM, Nyborg NCB, Strandgaard S, eds. Resistance Arteries, Structure and Function 1991. Elsevier Science Publishers B. V. 1991: 312 –315.
Rizzoni D, Porteri E, Castellano M, Bettoni G, Muiesan ML, Muiesan P, Giulini SM, Agabiti Rosei E. Vascular hypertrophy and remodeling in secondary hypertension. Hypertension. 1996; 28 (5): 785 –790.
Folkow B. Physiological aspects of primary hypertension. Physiol Rev. 1982; 62 : 347 –504.
Mulvany MJ, Aalkjaer C. Structure and function of small arteries. Physiol Rev. 1990; 70 : 921 –971.
Rizzoni D, Castellano M, Porteri E, Bettoni G, Muiesan ML, Agabiti-Rosei E. Vascular structural and functional alterations before and after the development of hypertension in SHR. Am J Hypertens. 1994; 7 : 193 –200.
Rizzoni D, Castellano M, Porteri E, Bettoni G, Muiesan ML, Agabiti Rosei E. Delayed development of hypertension after short-term nitrendipine treatment. Hypertension. 1994; 24 : 131 –139.
Rizzoni D, Castellano M, Porteri E, Bettoni G, Muiesan ML, Cinelli A, Agabiti Rosei E. Effects of low and high doses of fosinopril on the structure and function of resistance arteries. Hypertension. 1995; 26 : 118 –123.
Rizzoni D, Castellano M, Porteri E, Bettoni G, Muiesan ML, Cinelli A, Zulli R, Agabiti-Rosei E. Prolonged effects of short-term fosinopril on blood pressure and vascular morphology and function in rats. Am J Hypertens, In press.
Beschi M, Castellano M, Agabiti Rosei E, Rizzoni D, Rossini P, Muiesan G. Assessment of semi-automated catecholamine assay by HPLC: choice of reverse-phase C18 or cation-exchange columnns. Chromatographia. 1987; 24 : 455 –459.
Aalkjaer C, Heagerty AM, Petersen KK, Swales JD, Mulvany MJ. Evidence for increased media thickness, increased neural amine uptake, and depressed excitation-contraction coupling in isolated resistance vessels from essential hypertensives. Circ Res. 1987; 61 (2): 181 –186.
Mulvany MJ, Halpern W. Contractile properties of small resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res. 1977; 41 (1): 19 –26.
Mulvany MJ, Hansen PK, Aalkjaer C. Direct evidence that the greater contractility of resistance vessels in spontaneously hypertensive rats is associated with a narrowed lumen, a thickened media, and an increased number of smooth muscle cell layers. Circ Res. 1978; 43 (6): 854 –864.
Taddei S, Virdis A, Mattei P, Ghiadoni L, Sudano I, Salvetti A. Defective L-arginine-nitric oxide pathway in offspring of essential hypertensive patients. Circulation. 1996; 94 : 1298 –1303.
The SAMPLE Study Group. Ambulatory blood pressure is superior to clinic blood pressure in predicting treatment-induced regression of left ventricular hypertrophy. Circulation. 1997; 95 (6): 1464 –1470.
Clozel M, Kuhn H, Hefti F. Effects of angiotensin converting enzyme inhibitors and of hydralazine on endothelial function in hypertensive rats. Hypertension. 1990; 16 : 532 –540.
Schiffrin EL, Deng LY. Comparison of effects of angiotensin 1-converting enzyme inhibition and β-blockade for 2 years on function of small arteries from hypertensive patients. Hypertension. 1995; 25 (Part 2): 699 –703.